Simian virus 40 large T antigen binds to topoisomerase I

Simmons, D. T., Melendy, T., Usher, D., Stillman, B. (1996) Simian virus 40 large T antigen binds to topoisomerase I. Virology, 222 (2). pp. 365-74. ISSN 0042-6822 (Print)

URL: http://www.ncbi.nlm.nih.gov/pubmed/8806520
DOI: 10.1006/viro.1996.0433

Abstract

Binding of simian virus 40 (SV40) large T antigen to human and calf thymus topoisomerase I (topo I) was readily detected by using modified enzyme-linked immunosorbent assays and immunoblots. In addition to WT T antigen, binding could also be readily demonstrated with T antigen fragments from the amino-terminal region as well as with fragments missing this region, but much less so with small t antigen or with human p53. Antibody-blocking experiments showed that a monoclonal antibody that binds to the N-terminal region and several antibodies that recognize the central region of T antigen interfere with the binding to topo I. Our data are consistent with the existence of two separate topo I-binding regions in T antigen, one mapping within residues 82 to 246 and an apparently weaker one present after residue 246. By comparing the binding of T antigen to topo I with that of T antigen to DNA polymerase alpha or RPA, a single-stranded DNA-binding protein, it was determined that the T antigen-topo I interaction is much stronger and that the binding sites for topo I and DNA polymerase overlap, whereas the one for RPA differs. Several unwinding-defective mutants of T antigen were partially defective in their binding to topo I, suggesting that the binding to topo I is required for unwinding circular DNA. Finally, immunoprecipitation experiments demonstrated that T antigen can interact with DNA-bound topo I, indicating that such an interaction may take place during SV40 DNA replication.

Item Type: Paper
Uncontrolled Keywords: Animals Antibodies Monoclonal metabolism Antigens Polyomavirus Transforming genetics/*metabolism Cattle Cell Line DNA metabolism DNA Primase DNA Replication DNA Topoisomerases Type I genetics metabolism Humans Immunoblotting Point Mutation Protein Binding RNA Nucleotidyltransferases metabolism Rabbits Recombinant Fusion Proteins genetics metabolism Simian virus 40 metabolism Spodoptera cytology
Subjects: organism description > virus
CSHL Authors:
Communities: CSHL labs > Stillman lab
Highlight: Stillman, Bruce W.
Depositing User: CSHL Librarian
Date Deposited: 02 Mar 2012 15:53
Last Modified: 20 Jun 2017 19:38
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25055

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